The objective of the research is to compare transcriptionally active and inactive chromatin with respect to (a) initial yields of ionizing radiation-induced DNA damage and (b) repair capacity. Since the specific model system will be ribosomal chromatin, which contains the DNA coding for ribosomal RNA (rRNA), the work will be comparing the transcriptionally active ribosomal DNA characteristic of actively proliferating cells and the transcriptionally less active rDNA characteristic of quiescent (and generally more radio-resistant) cells. Rodent or human cell lines will be obtained during active exponential growth, during inhibition by nutrient or serum depletion, confluency, or drugs, or during stimulation with nutrients or insulin. The rate of ribosomal RNA synthesis and other metabolic parameters characteristic of proliferation rate (protein synthesis and phosphorylation, cyclic nucleotide levels) and radiation damage to and repair of rDNA will be determined. We have developed a system to analyze single-strand breaks, alkali-labile lesions, and enzyme-sensitive sites specifically in rDNA. The procedure involves releasing the rDNA from bulk nuclear DNA with a restriction endonuclease, treatment with a lesion-specific endonuclease, and sizing the rDNA fragments by agarose gel electrophoresis, then identifying rDNA by hybridization to rRNA. Furthermore, ribosomal chromatin from proliferating or plateau phase, irradiated or unirradiated cultures will be tested for its accessibility to other nucleases reported to be specific for structural features of active or inactive chromatin. Unlike the majority of nuclear DNA, the rRNA genes are a major class of DNA which is known to be differentially active in proliferating and plateau phase cells. Plateau phase cells have been considered a suitable two-dimensional model for the non- or slowly-growing and generally more radioresistant sub-populations of tumors. Thus, the results will help to define some critical areas of metabolism affected when nutrients or hormones stimulate the proliferation of plateau phase cells and enhance their radiosensitivity.